In humans, the endometrium, the uterine mucosal lining, undergoes dynamic changes throughout the menstrual cycle and pregnancy. Despite the importance of the endometrium as the site of implantation and nutritional support for the conceptus, there are no long-term culture systems that recapitulate endometrial function in vitro. We adapted conditions used to establish human adult stem-cell-derived organoid cultures to generate three-dimensional cultures of normal and decidualized human endometrium. These organoids expand long-term, are genetically stable and differentiate following treatment with reproductive hormones. Single cells from both endometrium and decidua can generate a fully functional organoid. Transcript analysis confirmed great similarity between organoids and the primary tissue of origin. On exposure to pregnancy signals, endometrial organoids develop characteristics of early pregnancy. We also derived organoids from malignant endometrium, and so provide a foundation to study common diseases, such as endometriosis and endometrial cancer, as well as the physiology of early gestation.
Infection of the bovine endometrium with Gram-negative bacteria commonly causes uterine disease. Toll-like receptor 4 (TLR4) on cells of the immune system bind Gram-negative bacterial lipopolysaccharide (LPS), stimulating the secretion of the proinflammatory cytokines interleukin 1B (IL1B) and IL6, and the chemokine IL8. Because the endometrium is the first barrier to infection of the uterus, the signaling cascade triggered by LPS and the subsequent expression of inflammatory mediators were investigated in endometrial epithelial and stromal cells, and the key pathways identified using short interfering RNA (siRNA) and biochemical inhibitors. Treatment of endometrial cells with ultrapure LPS stimulated an inflammatory response characterized by increased IL1B, IL6, and IL8 mRNA expression, and IL6 protein accumulation in epithelial cells, and by increased IL1B and IL8 mRNA expression, and IL6 and IL8 protein accumulation in stromal cells. Treatment of endometrial cells with LPS also induced the degradation of IKB and the nuclear translocation of NFKB, as well as rapid phosphorylation of mitogen-activated protein kinase 3/1 (MAPK3/1) and MAPK14. Knockdown of TLR4 or its signaling adaptor molecule, myeloid differentiation factor 88 (MYD88), using siRNA reduced the inflammatory response to LPS in epithelial and stromal cells. Biochemical inhibition of MAPK3/1, but not JNK or MAPK14, reduced LPS-induced IL1B, IL6, and IL8 expression in endometrial cells. In conclusion, epithelial and stromal cells have an intrinsic role in innate immune surveillance in the endometrium, and in the case of LPS this recognition occurs via TLR4- and MYD88-dependent cell signaling pathways.
MicroRNAs (miRNAs), a class of small noncoding RNAs that regulate gene expression, have fundamental roles in biological processes, including cell differentiation and proliferation. These small molecules mainly direct either target messenger RNA (mRNA) degradation or translational repression, thereby functioning as gene silencers. Epithelial cells of the uterine lumen and glands undergo cyclic changes under the influence of the sex steroid hormones estradiol-17beta and progesterone. Because the expression of miRNAs in human endometrium has been established, it is important to understand whether miRNAs have a physiological role in modulating the expression of hormonally induced genes. The studies herein establish concomitant differential miRNA and mRNA expression profiles of uterine epithelial cells purified from endometrial biopsy specimens in the late proliferative and midsecretory phases. Bioinformatics analysis of differentially expressed mRNAs revealed cell cycle regulation as the most significantly enriched pathway in the late proliferative-phase endometrial epithelium (P = 5.7 x 10(-15)). In addition, the WNT signaling pathway was enriched in the proliferative phase. The 12 miRNAs (MIR29B, MIR29C, MIR30B, MIR30D, MIR31, MIR193A-3P, MIR203, MIR204, MIR200C, MIR210, MIR582-5P, and MIR345) whose expression was significantly up-regulated in the midsecretory-phase samples were predicted to target many cell cycle genes. Consistent with the role of miRNAs in suppressing their target mRNA expression, the transcript abundance of predicted targets, including cyclins and cyclin-dependent kinases, as well as E2F3 (a known target of MIR210), was decreased. Thus, our findings suggest a role for miRNAs in down-regulating the expression of some cell cycle genes in the secretory-phase endometrial epithelium, thereby suppressing cell proliferation.
Objective To investigate the effects of testosterone and metformin on glucose metabolism in endometrium. Design In vitro experiment. Setting Reproductive and Genetic Center, Obstetrics and Gynecology, The First Hospital of Peking University, Beijing, China. Patient(s) Premenopausal women undergoing total abdominal hysterectomy for uterine myoma or adenomyosis. Intervention(s) Endometrial glandular epithelial cell culture. Main Outcome Measure(s) Insulin receptor substrate-1 (IRS-1) and glucose transporter 4 (GLUT-4) expression in endometrial glandular epithelial cell. Result(s) After the action of testosterone solution, the protein expressions of IRS-1mRNA, IRS-1, and GLUT-4 in endometrial glandular epithelial cells all decreased significantly. Metformin may reverse this effect. Conclusion(s) The high androgen levels in patients with polycystic ovary syndrome could induce insulin resistance in endometrial glandular epithelial cell, whereas metformin has an antagonizing effect.
Establishment of implantation in pig is accompanied by a coordinated interaction between the maternal uterine endometrium and conceptus development. We investigated the expression profiles of endometrial tissue on Days 9, 12 and 15 of pregnancy and on Day 12 of non-pregnancy in Yorkshire, and performed a comprehensive analysis of long non-coding RNAs (lncRNAs) in endometrial tissue samples by using RNA sequencing. As a result, 2805 novel lncRNAs, 2,376 ( 301 lncRNA and 2075 mRNA) differentially expressed genes (DEGs) and 2149 novel transcripts were obtained by pairwise comparison. In agreement with previous reports, lncRNAs shared similar characteristics, such as shorter in length, lower in exon number, lower at expression level and less conserved than protein coding transcripts. Bioinformatics analysis showed that DEGs were involved in protein binding, cellular process, immune system process and enriched in focal adhesion, Jak-STAT, FoxO and MAPK signaling pathway. We also found that lncRNAs TCONS_01729386 and TCONS_01325501 may play a vital role in embryo preimplantation. Furthermore, the expression of FGF7, NMB, COL5A3, S100A8 and PPP1R3D genes were significantly up-regulated at the time of maternal recognition of pregnancy (Day 12 of pregnancy). Our results first identified the characterization and expression profile of lncRNAs in pig endometrium during pre-implantation phases.
Matrix metalloproteinases (MMPs) are key enzymes involved in extracellular matrix remodelling. In the human endometrium, the expression and activity of several MMPs are maximal during the menstrual phase. Moreover, MMPs are thought to be involved in the pathogenesis of endometriosis and cancers, in particular with invasion and metastasis. We recently reported that MMP-27 is a unique MMP with an intracellular retention motif. We investigated the expression and cellular localization of MMP-27 in the cycling human endometrium and in endometriotic lesions. MMP-27 mRNA was detected throughout the menstrual cycle. Despite large interpatient variations, mRNA levels increased from the proliferative to the secretory phase, to peak during the menstrual phase. MMP-27 was immunolocalized in large isolated cells scattered throughout the stroma and around blood vessels: these cells were most abundant at menstruation and were identified by immunofluorescence as CD45(+), CD163(+) and CD206(+) macrophages. CD163(+) macrophages were also abundant in endometriotic lesions, but showed different patterns in ovarian or peritoneal endometriotic lesions (co-labelling for CD206 and MMP-27) and rectovaginal lesions (no co-labelling). In conclusion, MMP-27 is expressed in a subset of endometrial macrophages related to menstruation and in ovarian and peritoneal endometriotic lesions.
The endometrium is a complex tissue that lines the inside of the endometrial cavity. The gene expression of the different endometrial cell types is regulated by ovarian steroids and paracrine-secreted molecules from neighbouring cells. Due to this regulation, the endometrium goes through cyclic modifications which can be divided simply into the proliferative phase, the secretory phase and the menstrual phase. Successful embryo implantation depends on three factors: embryo quality, the endometrium's state of receptivity, and a synchronised dialogue between the maternal tissue and the blastocyst. There is a need to characterise the endometrium's state of receptivity in order to prevent reproductive failure. No single molecular or histological marker for this status has yet been found. Here, we review the global transcriptomic analyses performed in the last decade on a normal human endometrium. These studies provide us with a clue about what global gene expression can be expected for a non-pathological endometrium. These studies have shown endometrial phase-specific transcriptomic profiles and common temporal gene expression patterns. We summarise the biological processes and genes regulated in the different phases of natural cycles and present other works on different conditions as well as a receptivity diagnostic tool based on a specific gene set profile. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure. ► We review the transcriptomic data on normal human endometrium. ► Single molecule markers cannot classify the status of the endometrium. ► Transcriptomic profiles have proven efficient to classify the endometrial status.
In ruminants, both the endometrium and the conceptus (embryo and associated extraembryonic membranes) trophectoderm synthesizes and secretes prostaglandins (PG) during early pregnancy. In mice and humans, PGs regulate endometrial function and conceptus implantation. In Study One, bred ewes received intrauterine infusions of vehicle as a control (CX) or meloxicam (MEL), a PG synthase (PTGS) inhibitor from Days 814 postmating, and the uterine lumen was flushed on Day 14 to recover conceptuses and assess their morphology. Elongating and filamentous conceptuses (12 cm to >14 cm in length) were recovered from all CX-treated ewes. In contrast, MEL-treated ewes contained mostly ovoid or tubular conceptuses. PTGS activity in the uterine endometrium and amounts of PGs were substantially lower in uterine flushings from MEL-treated ewes. Receptors for PGE2 and PGF2 alpha were present in both the conceptus and the endometrium, particularly the epithelia. In Study Two, cyclic ewes received intrauterine infusions of CX, MEL, recombinant ovine interferon tau (IFNT), or IFNT and MEL from Days 10-14 postestrus. Infusion of MEL decreased PGs in the uterine lumen and expression of a number of progesterone-induced endometrial genes, particularly IGFBP1 and HSD11B1. IFNT increased endometrial PTGS activity and the amount of PGs in the uterine lumen. Interestingly, IFNT stimulation of many genes (FGF2, ISG15, RSAD2, CST3, CTSL, GRP, LGALS15, IGFBP1, SLC2A1, SLC5A1, SLC7A2) was reduced by co-infusion with MEL. Thus, PGs are important regulators of conceptus elongation and mediators of endometrial responses to progesterone and IFNT in the ovine uterus.
Objective To investigate whether G protein-coupled estrogen receptor (GPER, also known as GPR30 and GPER1) stabilizes hypoxia-inducible factor 1α (HIF-1α) in eutopic endometrium (EuEM) of endometriosis. Design Immunohistochemical analysis and experimental in vitro study. Setting University hospital. Patient(s) Patients with or without endometriosis. Intervention(s) The EuEM and normal control endometrium (CoEM) were obtained by curettage. Primary cultured endometrial stromal cells (ESCs) were treated with 17β-E2 , G1, or G15. Main Outcome Measure(s) The EuEM and CoEM were collected for immunohistochemistry. Western blot, polymerase chain reaction, ELISA, and dual luciferase experiments were used to detect expression of GPER, HIF-1α, vascular endothelial growth factor (VEGF), and matrix metalloproteinase 9 (MMP9) in ESCs. Estradiol and G1 were used as agonists of GPER, G15 as an antagonist. Migration of ESCs and endothelial tube formation of human umbilical vein endothelial cells cultured in medium collected from ESCs were measured. Result(s) Protein levels of GPER and HIF-1α were higher in EuEM than in CoEM. Protein levels of HIF-1α but not HIF-1α mRNA levels increased concurrently with GPER after E2 and G1 treatment. Furthermore, expression and activity of VEGF and MMP9 increased under E2 and G1 stimulation. However, these effects disappeared when GPER was blocked. Conclusion(s) G protein-coupled estrogen receptor stabilizes HIF-1α and thus promotes HIF-1α–induced VEGF and MMP9 in ESCs, which play critical roles in endometriosis.
The steroid hormone progesterone (P 4 ) plays a key role in the reproductive events associated with pregnancy establishment and maintenance. High concentrations of circulating P 4 in the immediate post-conception period have been associated with an advancement of conceptus elongation, an associated increase in interferon-τ production and higher pregnancy rates in cattle. Using in vitro and in vivo models and ∼8500 bovine oocytes across six experiments, the aim of this study was to establish the route through which P 4 affects bovine embryo development in vitro and in vivo . mRNA for P 4 receptors was present at all stages of embryo development raising the possibility of a direct effect of P 4 on the embryo. Exposure to P 4 in vitro in the absence or presence of oviduct epithelial cells did not affect the proportion of embryos developing to the blastocyst stage, blastocyst cell number or the relative abundance of selected transcripts in the blastocyst. Furthermore, exposure to P 4 in vitro did not affect post-hatching elongation of the embryo following transfer to synchronized recipients and recovery on Day 14. By contrast, transfer of in vitro derived blastocysts to a uterine environment previously primed by elevated P 4 resulted in a fourfold increase in conceptus length on Day 14. These data provide clear evidence to support the hypothesis that P 4 -induced changes in the uterine environment are responsible for the advancement in conceptus elongation reported previously in cattle and that, interestingly, the embryo does not need to be present during the period of high P 4 in order to exhibit advanced elongation.